Extraction of nucleic acid

ABSTRACT

Methods of obtaining a sample of target nucleic acid from cells containing the target nucleic acid and genomic DNA or RNA are disclosed. In contrast to prior art protocols, this method does not require the cells containing the target nucleic acid to be lysed and instead is based on the observation when cells are suspended in an aqueous medium and the target nucleic acid are released into the medium through the cell walls. The invention therefore helps to avoid the use of cell lysis, heating, extremes of pH, water immiscible solvents, and electrical fields used in prior art nucleic acid extraction methods. The present invention is particularly applicable to the separation of non-genomic nucleic acid, such as cellular vector DNA or RNA, self-replicating satellite nucleic acids or plasmid DNA, from genomic nucleic acids, such as host cell chromosomes and ribosomal RNA.

FIELD OF THE INVENTION

The present invention relates to nucleic acid purification, and inparticular to a method of obtaining a sample of target nucleic acid fromcells containing the target nucleic acid and genomic DNA or RNA.

BACKGROUND OF THE INVENTION

Current methods of purifying non-genomic nucleic acids, such as vectorDNA or RNA, rely on extensive cell lysis or cell wall degradation torelease the nucleic acid present in the cells, using cell lysing enzymessuch as lysozyme, chaotropic agents or extremes of pH and heat (see forexample the methods disclosed in Maniatis, ‘Molecular Cloning, ALaboratory Manual’, Book 1, Sections 1.21-1.23, Cold Spring HarborPress, 1989). However, while this approach releases the non-genomicnucleic acid from the cells, it is accompanied by the majority of theother host genomic nucleic acid contained in the cell, and otherimpurities such as cellular endotoxins. This necessitates a sequence ofdifficult and time consuming purification steps in order purify targetnucleic acid molecules from these impurities. These steps are needed asthe release of RNA or host chromosomal DNA can then interfere with thedownstream analysis or functionality of the target nucleic acid which,if present in the sample of the target nucleic acid. Further, extensivecell lysis often results in the generation of a viscous mass of cellularmaterial making further purification difficult.

Calvin and Hanawalt (J. Bacteriol, 170(6): 2796-2801, 1988) disclosesthe use of reverse electroporation for the recovery of plasmid DNA fromE. coli. While plasmids were recovered in the method, most of theconditions tested also led to the extraction of cellular DNA and RNA. Inorder to obtain the plasmids, electrical field strengths of13,000-15,000 V/cm were required.

WO 00/29563 (Cambridge Molecular Technologies Limited) discloses amethod of separating plasmid from genomic DNA employing a waterimmiscible organic solvent, a chaotrope and heating to at least 65° C.This led to the extraction of the plasmid DNA into the organic phase.

EP 0 657 530 A (Gen-Probe Inc) discloses a method of extracting nucleicacid from bacterial cells employing a permeabilising reagent comprisinga non-ionic detergent and a metal chelating agent, such as EDTA, incombination with heating to 80-100° C.

An additional problem is that the procedures to purify a sample of thetarget nucleic acid from the impurities typically requires severalcentrifugation steps and during the purification procedures, the volumeof the sample changes significantly. These factors mean that existingpurification protocols need to be carried out by a technician and arenot easily automated.

SUMMARY OF THE INVENTION

Broadly, the present invention provides method of obtaining a sample oftarget nucleic acid from cells containing the target nucleic acid andgenomic DNA or RNA. In contrast to prior art protocols, this method doesnot require the cells containing the target nucleic acid to be lysed andinstead relies on the observation that after separation from culturebroth or medium, cells can be resuspended in an aqueous medium and thetarget nucleic acid released into the aqueous medium through the cellwalls without the need for cell lysis. The present invention may inpreferred embodiments avoid the heating used in many prior art nucleicacid extraction methods. The present invention is particularlyapplicable to the separation of non-genomic nucleic acid, such ascellular vector DNA or RNA, self-replicating satellite nucleic acids orplasmid DNA, from genomic nucleic acids, such as host cell chromosomesand ribosomal RNA.

Accordingly, in a first aspect, the present invention provides a methodof obtaining a sample of target nucleic acid from cells containing thetarget nucleic acid and genomic nucleic acid, the method comprising thesteps of:

-   -   separating the cells from culture broth;    -   suspending the cells in an aqueous medium which causes the        target nucleic acid to leak from the cells into the aqueous        medium; and    -   obtaining the sample of the nucleic acid from the aqueous        medium;    -   wherein the cells are substantially not lysed during the above        steps and substantially retain the genomic nucleic acid within        the cells

Preferably, the method does not substantially cause the release ofcellular endotoxins, thereby allowing the separation of the targetnucleic acid from the cellular endotoxins, in addition to genomicnucleic acid or RNA.

While not wishing to be bound by any particular theory, the inventorsbelieve that the change in the environment of the cells from theconditions in culture broth to the conditions in the aqueous medium,typically at lower ionic strength, makes the cell walls ‘leaky’ orslightly porous to the target nucleic acid, and especially nucleic acidsuch as vectors. This allows the target nucleic acid to diffuse into theaqueous medium without the need to lyse the cells, e.g. by contactingthe cells with lysozyme, chaotropes or extremes of pH or heat, with theresult that the cells retain impurities such as genomic DNA and cellularproteins, thereby avoiding the need for complicated procedures to removethese materials from a sample comprising the impurities and the targetnucleic acid. In a preferred embodiment of the invention, the targetnucleic acids may be 100 kb or less, or more preferably 50 kb or less,or more preferably 20 kb or less or even more preferably 10 kb or lessin size. The size of nucleic acids can be determined by those skilled inthe art, e.g. using gel electrophoresis technique employing apolyacrylamide or agarose gel, e.g. see Ausubel et al, ‘Short Protocolsin Molecular Biology’, John Wiley and Sons, NY, 1992.

In the present invention, “not substantially lysed” means preferablyless than 20%, more preferably less than 10%, more preferably less than5%, more preferably less than 2% and most preferably less than 1% of thecells in the population treated according to the method are lysed. Theextent of cell lysis can readily be determined, e.g. by counting lysedand non-lysed cells present in a sample under a microscope.

Preferably, the target nucleic acid is non-genomic nucleic acid which isseparated from genomic nucleic acid retained inside the cells.Non-genomic nucleic acid includes vectors, plasmids, self replicatingsatellite nucleic acid or cosmid DNA, or vector RNA. Other forms oftarget nucleic acids may include bacteriophages such as Lambda, M13 andviral nucleic acids. In a preferred embodiment, the non-genomic nucleicacid sample is plasmid DNA.

The method is widely applicable to many different types of host cells.For example Gram negative and Gram positive bacteria, filamentousbacteria or fungi such as Streptomyces, yeast cells, plant cells andplant protoplasts. The use of gram negative bacteria is preferred, suchas E. Coli strains, e.g. strains JM109 or HB101.

Typically, the cells are treated at ambient temperatures between 0 and90° C. Unlike many of the prior art methods, preferred embodiments ofthe present invention may employ temperatures less than about 60° C.,and more preferably less than about 40° C. Preferred temperature rangesemployed in the method of the invention are between 0 and 60° C.,between 0 and 40° C. and most preferably between 15 and 40° C.

The present invention includes the use of a variety of differentconditions to cause the release of target nucleic acid from the cellsinto the surrounding aqueous medium. Examples of preferred aqueous mediainclude water, salts such as NaCl, in hypotonic or hypertonicconditions. Examples of low salt buffers include Tris. HCl, e.g. at pH6-9, optionally including EDTA, a potassium salt such as potassiumacetate, optionally including KCl, or a divalent or trivalent metal saltsuch as CaCl₂, preferably at a concentration between 10 and 250 mM, andmore preferably between 50 and 150 mM.

Alternatively or additionally, a sugar solution can also be employed,preferably at concentrations between 0.05 and 1.0M, and more preferablybetween 0.1 and 0.5M. A preferred sugar solution is a sucrose solution.

Optionally, the aqueous media may additionally include a proteinase suchas Proteinase K to improve the yield of the target nucleic acid. Inparticular, the inventors have found that including a proteinase withthe divalent or trivalent metal ion salt greatly increases the leakageof the target nucleic acid from the cells. The use of Proteinase K andCaCl₂ is particularly preferred.

In a preferred embodiment, the aqueous medium is a low salt buffer.Preferably, the low salt buffer comprises Tris HCl, optionally incombination with EDTA. In this case, preferably the low salt buffercomprises 5 mM to 50 mM Tris HCl and most preferably about 10 mM TrisHCl. Where EDTA is present, it is preferably present at a concentrationbetween 0.1 mM and 100 mM, more preferably at a concentration between0.1 and 50 mM, and most preferably at a concentration of about 1 mM.

In another preferred embodiment, the low salt buffer is a potassium saltat a concentration between 10 mM and 30 mM, most preferably at aconcentration of about 16 mM. A preferred pH of potassium salt solutionsis about pH 4.

Preferably, the aqueous medium has a pH between pH 3 and 11, morepreferably between pH 6 and 9, and most preferably a pH of about 8.5.

Slightly higher salt concentrations can also be used, e.g. by employingaqueous media including sodium chloride, e.g. at a concentration whichis preferably between about 50 mM and 250 mM, and more preferably about150 mM. A preferred pH of sodium chloride solutions is about pH 7. Thesalts solutions or aqueous media may be buffered with standardlaboratory buffers such as biological buffers, e.g. MES, MOPS, HEPES orAcetate buffers or even phosphate buffers such as PBS.

The aqueous medium may comprise a detergent, either alone or incombination with one or more of the other components described herein.It is preferred that the detergent is a non-ionic detergent such asTween 20 that do not inhibit subsequent use of the target nucleic acid,e.g. in analytical techniques. The aqueous medium may further comprise aRNA nuclease or a DNA nuclease to selectively degrade unwanted RNA orDNA targets from a mixture released into the surrounding medium.Additionally, a protease may be employed to degrade any releasedproteins.

The present invention makes it possible to avoid the harsh conditionsused in many prior art methods for purifying nucleic acid. Whenconditions are made harsher, the cell wall degrades extensively and aconsiderable amount of unwanted nucleic acids are released. Examples ofharsh conditions include, but are not limited to, 0.1M NaOH or 0.1M NaOHwith 1% SDS. In some cases, harsh conditions include the use oftemperatures of 65° C. or above and/or the use of water immiscibleorganic solvents and/or the use of electroporation. Thus, the method ofthe invention is preferably carried out in the absence of an electricalfield capable of causing poration, e.g. having a field strength above10,000 V/cm.

In some embodiments, the method may involve one or more of theadditional steps of:

-   -   (a) isolating the target nucleic acid; or    -   (b) analysing the target nucleic acid; or    -   (c) amplifying the target nucleic acid; or    -   (d) sequencing the target nucleic acid.

These steps are discussed in more detail below.

In a further embodiment of the invention, the target nucleic acid, suchas a plasmid, can be separated from the cells according to the presentinvention and the resulting aqueous media, i.e. the supernatant, useddirectly with out the requirement for further purification steps, e.g.for PCR or other analytical methods.

A range of techniques are available to the skilled person for purifyingnucleic acid that has leaked from cells and is present in the aqueousmedia. Examples of purification techniques include ion-exchange,electrophoresis, silica solid phase with chaotropic salt extraction,precipitation, flocculation, filtration, gel filtration, centrifugation,alcohol precipitation and/or the use of a charge switch materialdescribed in our copending applications European Patent Application No:98957019.7, U.S. patent application Ser. No. 09/736,632 and WO 02/48164and other purification or separation methods well known in the art. Inpreferred embodiments, the target nucleic acid is purified using acharge switch material, e.g. present on a solid phase, a pipette tip,beads (especially magnetic beads), a porous membrane, a frit, a sinter,a probe or dipstick, a tube (PCR tube, Eppendorf tube) or a microarray.Charge switch materials may be solid phases or soluble. They compriseionisable groups such that they are capable of binding nucleic acid at afirst pH (typically between pH 5.0 and 9.0), and then releasing it at asecond higher pH (typically less than 10.5). Examples of solid phasecharge switch materials are those in which (1) the ionisable groups areseparately immobilised on a solid support by covalent or ionic bondingor by adsorption, (2) the ionisable groups are separately attached to apolymer, said polymer being immobilised on a solid support by covalentor ionic bonding or by adsorption, (3) the ionisable groups arepolymerised, optionally by means of cross-linking reagents, and thepolymer is immobilised on a solid support by covalent or ionic bondingor by adsorption. By way of example, the charged groups in the chargeswitch material may be provided by a biological buffer (e.g. Tris,Bis-Tris, polyTris or poly Bis-Tris), a polyhydroxylated amine, adetergent or surfactant, a carbohydrate, a nucleic acid base, aheterocyclic nitrogen-containing compound, a monoamine, a biologicaldye, or a negatively ionisable group, the pKa of which is between about3.0 and 7.0 and a metal oxide which is positively charged at said firstpH, and optionally also at said second pH.

The target nucleic acid may also be the subject of amplification,conveniently using the polymerase chain reaction. PCR techniques for theamplification of nucleic acid are described in U.S. Pat. No. 4,683,195.In general, such techniques require that sequence information from theends of the target sequence is known to allow suitable forward andreverse oligonucleotide primers to be designed to be identical orsimilar to the polynucleotlde sequence that is the target for theamplification. PCR comprises steps of denaturation of template nucleicacid (if double-stranded), annealing of primer to target, andpolymerisation. The nucleic acid probed or used as template in theamplification reaction may be genomic DNA, cDNA or RNA. PCR can be usedto amplify specific sequences from genomic DNA, specific RNA sequencesand cDNA transcribed from mRNA, bacteriophage or plasmid sequences.References for the general use of PCR techniques include Mullis et al,Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCRTechnology, Stockton Press, NY, 1989, Ehrlich et al, Science,252:1643-1650, (1991), “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, Academic Press, New York, (1990).

In some embodiments, the method may involve one or more initial stepssuch as:

-   -   culturing the cells in the culture broth or growing on selective        media on plates. The cells/colonies are separated and then        placed in the aqueous medium. Separation may be achieved by        centrifugation, filtration, magnetic bead separation, or using a        probes or dipsticks.

Thus, in one embodiment, the method comprises growing the cells in brothor culture medium, separating the cells and resuspending them in theaqueous media to cause the cells to become leaky and release the targetnucleic acid.

Preferred embodiments of the present invention will now be described inmore detail, by way of example and not limitation, with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 5 show the plasmid DNA samples purified according to thepresent invention run on agarose gels stained with ethidium bromide. Thefigures are labelled with the conditions (reagents and concentrations)used for each purification. The nucleic acid bands are all from plasmidDNA, in supercoiled, nicked or linear forms. No bands were observed forgenomic DNA, demonstrating the selectivity of the method of theinvention.

EXAMPLE 1

An overnight 5 ml culture of JM109 containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of10 mM Tris. HCl, 10 mM EDTA pH 8.5. Following a 5 minute incubation, thecells were centrifuged leaving a clear supernatant containing theplasmid. A sample was taken for PCR analysis and the remaining plasmidwas purified by adjusting the pH to 4 with 166 μl of a 1.6M potassiumacetate buffer and then adding 1 mg of magnetic beads derivatised withpositively charged groups. The magnetic beads were incubated for 1minute to bind the plasmid, separated on a magnet, washed twice withwater and the DNA recovered by eluting with 100 μl of 10 mM Tris.HCl pH8.5. Plasmid purity was confirmed by gel electrophoresis and identityconfirmed by PCR and sequencing.

EXAMPLE 2

An overnight 5 ml culture of XL-1 Blue containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of10 mM Tris. HCl, 10 mM EDTA pH 8.5. Following a 5 minute incubation, thecells were centrifuged leaving a clear supernatant containing theplasmid. A sample was taken for PCR analysis and the remaining plasmidwas purified by adjusting the pH to 4 with 166 μl of a 1.6M potassiumacetate buffer and then adding 1 mg of magnetic beads derivatised withpositively charged groups. The magnetic beads were incubated for 1minute to bind the plasmid, separated on a magnet, washed twice withwater and the DNA recovered by eluting with 100 ul of 10 mM Tris.HCl pH8.5. Plasmid purity was confirmed by gel electrophoresis and identityconfirmed by PCR and sequencing.

EXAMPLE 3

An overnight 5 ml culture of XL-1 Blue containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl ofwater. Following a 5 minute incubation, the cells were centrifugedleaving a clear supernatant containing the plasmid. Plasmid purity wasconfirmed by gel electrophoresis and identity confirmed by PCR.

EXAMPLE 4

An overnight 5 ml culture of XL-1 Blue containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of0.15M NaCl. Following a 5 minute incubation, the cells were centrifugedleaving a clear supernatant containing the plasmid. Plasmid purity wasconfirmed by gel electrophoresis and identity confirmed by PCR.

EXAMPLE 5

An overnight 5 ml culture of XL-1 Blue containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of10 mM potassium acetate, 10 mM potassium chloride pH 4. Following a 5minute incubation, the cells were centrifuged leaving a clearsupernatant containing the plasmid. Plasmid purity was confirmed by gelelectrophoresis and identity confirmed by PCR.

EXAMPLE 6

An overnight 5 ml culture of XL-1 Blue containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of10 mM Tris HCl, 1% Tween 20. Following a 5 minute incubation, the cellswere centrifuged leaving a clear supernatant containing the plasmid.Plasmid purity was confirmed by gel electrophoresis and identityconfirmed by PCR.

EXAMPLE 7

An overnight 5 ml culture of XL-1 Blue containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of10 mM Tris HCl, 1% Tween 20. Following a 5 minute incubation, the cellsand supernatant containing the plasmid were added to a PCR reaction fordirect analysis.

EXAMPLE 8

An overnight 5 ml culture of JM109 containing pUC19 plasmid wascentrifuged to pellet the cells which were then resuspended in 500 μl of10 mM Tris. HCl, 10 mM EDTA pH 8.5 plus RNase A at 20 ug/ml. Following a5 minute incubation, the cells were centrifuged leaving a clearsupernatant containing the plasmid. A sample was taken for PCR analysisand the remaining plasmid was purified by adjusting the pH to 4 with 166μl of a 1.6M potassium acetate buffer and then adding 1 mg of magneticbeads derivatised with positively charged groups. The magnetic beadswere incubated for 1 minute to bind the plasmid, separated on a magnet,washed twice with water and the DNA recovered by eluting with 100 μl of10 mM Tris.HCl pH 8.5. Plasmid purity was confirmed by gelelectrophoresis

EXAMPLE 9

A solution of 10 mM Tris HCl, 10 mM EDTA, pH 8.5 was diluted with waterto the following percentage concentrations (%): 100, 20, 15, 10 and 5.Then, 5×1.5 ml cultures from 200 ml pUC19/XL1-Blue overnight culturewere spun down to pellet the cells. The supernatant was removed. About500 μls of the range of buffer concentrations were added to the 5 tubeswith 5 μl Proteinase K (20 mg/ml) and 5 μl RNase A (5 mg/ml). The 5samples were fully resuspended and incubated at room temperature for 15mins. After 15 mins, the samples were spun down again to pellet thecells. The supernatant was collected to a new tube and the pellet wasdiscarded. To each tube, 500 μl of a 1.5 M potassium acetate buffer pH 4(PB) with 40 μl of charge switch magnetic beads were added and the tubeswere fully resuspended, incubated for 1 min, separated on a magneticrack, washed twice and eluted in 50 μl Elution Buffer (EB). 10 μl ofeach sample was run on a 1% agarose gel stained with ethidium bromide asshown in FIG. 1.

EXAMPLE 10

Sucrose was made up to the following concentrations (M): 0.5, 0.4, 0.3,0.2 and 0.1. 5×1.5 ml cultures from a 200 ml pUC19/XL1-Blue overnightculture were spun down to pellet the cells. The supernatant was removed.500 μls of the range of sucrose concentrations (M) were added to the 5tubes. 5 μl Proteinase K and 5 μl RNase A was added to each tube asbefore. The 5 samples were fully resuspended and incubated at roomtemperature for 15 mins. After 15 mins, the samples were spun down againto pellet the cells. The supernatant was collected to a new tube and thepellet was discarded. 500 μl of PB and 40 μl of CST magnetic beads wereadded and the tubes were fully resuspended, incubated for 1 min,separated on a magnetic rack, washed twice and eluted in 50 μl ElutionBuffer (EB). 10 μl of each sample was run on a 1% agarose gel stainedwith ethidium bromide, as shown in FIG. 2.

EXAMPLE 11

CaCl₂ was made up to the following concentrations (mM): 200, 175, 150,125 and 100. Five×1.5 ml cultures from a 200 μl pUC19/XL1-Blue overnightculture were spun down to pellet the cells. The supernatant was removed.About 500 μls of the range of CaCl₂ concentrations were added to the 5tubes. 5 μl Proteinase K and 5 μl RNase A was added to each tube asbefore. The 5 samples were fully resuspended and incubated at roomtemperature for 15 mins. After 15 mins, the samples were spun down againto pellet the cells. The supernatant was collected to a new tube and thepellet was discarded. 500 μl of PB and 40 μl of charge switch magneticbeads were added and the tubes were fully resuspended, incubated for 1min, separated on a magnetic rack, washed twice and eluted in 50 μlElution Buffer (EB). 10 μl of each sample was run on a 1% agarose gelstained with ethidium bromide, as show in FIG. 3.

EXAMPLE 12

To confirm that the leaked DNA is pUC19 vector plasmid, 4 PCR reactionswere set up using pUC19 vector primers to amplify a 700 bp PCR fragment.2 were set up using DNA from a leaky cell extraction and 2 were set upusing a sample of pUC19 from laboratory vector stocks. 10 μls of eachproduct was run on an ethidium bromide stained 1% agarose gel with a 1kb extension ladder, see FIG. 4.

EXAMPLE 13

Nine×1.5 ml cultures from a 200 μl pUC19/XL1-Blue overnight culture werespun down to pellet the cells. The supernatant was removed. Thefollowing range of 100 mM CaCl₂ volumes (μl) were added to the 9samples: 25, 50, 100, 200, 300, 400, 500, 600 and 700. 5 μl Proteinase Kand 5 μl RNase A was added to each tube as before. The 9 samples werefully resuspended and incubated at room temperature for 15 mins. After15 mins, the samples were spun down again to pellet the cells. Thesupernatant was collected to a new tube and the pellet was discarded.500 μl of precipitation buffer and 40 μl of CST magnetic beads wereadded and the tubes were fully resuspended, incubated for 1 min,separated on a magnetic rack, washed twice and eluted in 50 μl ElutionBuffer (EB). 10 μl of each sample was run on a 1% agarose gel stainedwith ethidium bromide, see FIG. 5.

The references cited herein are all expressly incorporated by referencein their entirety.

1. A method of obtaining a sample of target nucleic acid from cellscontaining the target nucleic acid and genomic nucleic acid, the methodcomprising the steps of: separating the cells from culture broth;suspending the cells in an aqueous medium which causes the targetnucleic acid to leak from the cells into the aqueous medium; andobtaining the sample of the nucleic acid from the aqueous medium;wherein the cells are substantially not lysed during the above steps andsubstantially retain the genomic nucleic acid within the cells.
 2. Themethod of claim 1, wherein the method is carried out at a temperature ofless than 60° C. and in the absence of an electrical field capable ofcausing cell poration.
 3. The method of claim 2, wherein the method iscarried out a temperature of less than 40° C.
 4. The method of claim 1,wherein cellular proteins are substantially retained within the cells.5. The method of claim 1, wherein the target nucleic acid is non-genomicnucleic acid.
 6. The method of claim 5, wherein the genomic nucleic acidis host cell chromosomal DNA or ribosomal RNA.
 7. The method of claim 1,wherein the target nucleic acid sample is a vector, a plasmid, satelliteor cosmid DNA or vector RNA.
 8. The method of claim 7, wherein thetarget nucleic acid sample is plasmid DNA.
 9. The method of claim 1,wherein the cells are a gram negative microorganism.
 10. The method ofclaim 9, wherein the cells are E. coli.
 11. The method of claim 1,wherein the aqueous medium is water, a low salt buffer, a salt solution,or a sugar solution.
 12. The method of claim 1, wherein the aqueousmedium has a pH between pH 6 and
 9. 13. The method of claim 11, whereinthe aqueous medium is a low salt buffer comprising Tris. HCl.
 14. Themethod of claim 13, wherein the Tris. HCl buffer has a concentrationbetween 5 mM and 50 mM.
 15. The method of claim 13, wherein the Tris.HCl buffer further comprises EDTA.
 16. The method of claim 13, whereinthe Tris. HCl buffer has a pH of about 8.5.
 17. The method of claim 11,wherein the aqueous medium is a low salt buffer comprising potassiumacetate/KCl.
 18. The method of claim 17, wherein the potassiumacetate/KCl has a concentration between 10 mM and 30 mM.
 19. The methodof claim 17, wherein the potassium acetate/KCl has a pH of about
 4. 20.The method of claim 11, wherein the aqueous medium is a salt solution.21. (Cancelled)
 22. (Cancelled)
 23. (Cancelled)
 24. The method of claim11, wherein the aqueous medium is a sugar solution.
 25. The method ofclaim 1, wherein the aqueous medium further comprises a proteinase. 26.The method of claim 25, wherein the proteinase is Proteinase K.
 27. Themethod of claim 1, wherein the aqueous medium further comprises adetergent.
 28. The method of claim 27, wherein the detergent is anon-ionic detergent.
 29. The method of claim 1, wherein the aqueousmedium further comprises a RNA nuclease, a DNA nuclease a protease or acombination of two or more of said enzymes.
 30. The method of claim 1,further comprising purifying the target nucleic acid present in thesample of target nucleic acid.
 31. The method of claim 1, furthercomprising isolating the sample of the target nucleic acid.
 32. Themethod of claim 1, further comprising analysing the sample of the targetnucleic acid.
 33. The method of claim 1, further comprising amplifyingthe sample of the target nucleic acid.
 34. The method of claim 1,further comprising sequencing the sample of the target nucleic acid. 35.The method of claim 31, wherein the isolation of the target nucleic acidis by ion-exchange, electrophoresis, silica solid phase extraction,precipitation, flocculation, filtration, gel filtration, centrifugation,alcohol precipitation and the use of a charge switch material.
 36. Themethod of claim 20, wherein said salt solution is a sodium chloridesolution.
 37. The method of claim 36, wherein the sodium chloridesolution has a concentration between about 50 mM and 250 mM and/or a pHof about pH
 7. 38. The method of claim 20, wherein said salt solution isa divalent or trivalent metal salt solution.
 39. The method of claim 38,wherein the aqueous medium is a divalent metal ion solution.
 40. Themethod of claim 39 wherein the divalent metal in solution is a CaCl₂solution.
 41. The method of claim 40, wherein the CaCl₂ solution is at aconcentration between 10 and 250 mM.
 42. The method of claim 24 whereinthe sugar solution is a sucrose solution having a concentration between0.05 and 1.0M.
 43. The method of claim 31, wherein the isolation of thetarget nucleic acid is by ion-exchange, electrophoresis, silica solidphase extraction, precipitation, flocculation, filtration, gelfiltration, centrifugation, alcohol precipitation or the use of a chargeswitch material.